Systems and methods for genotyping seed components

ABSTRACT

The invention provides methods for obtaining genetic material from plant embryos while preserving their viability as well as methods for performing a molecular analysis of plant embryos, particularly with small quantities of genetic material. The methods may include the steps of collecting shed cellular material from one or more embryos; obtaining DNA from the shed cellular material; performing a molecular analysis of the DNA; and germinating at least one of the one or more embryos. A further extension of this method includes determining whether to germinate and grow the embryo or to discard the embryo based on its genotype as part of a breeding process.

BACKGROUND OF THE INVENTION

It is conventional practice in plant breeding to grow plants from seedof known parentage. Seed are planted in experimental plots, growthchambers, greenhouses, or other growing conditions and plants arisingfrom the seed are either cross pollinated with other plants of knownparentage or self-pollinated. The resulting seed are the offspring ofthe two parent plants or the self-pollinated plant, and are harvested,processed and planted to continue the plant breeding cycle. Specificlaboratory or field-based tests may be performed on the plants, planttissues, seed or seed tissues, in order to aid in the breeding process.

Generations of plants based on known crosses or self-pollinations areplanted and then tested to see if these lines or varieties are movingtowards characteristics that are desirable in the marketplace. Examplesof desirable traits include, but are not limited to, increased yield,increased homozygosity, improved or newly conferred resistance and/ortolerance to specific herbicides and/or pests and pathogens, increasedoil content, altered starch content, nutraceutical composition, droughttolerance, and specific morphological based trait enhancements.

As can be appreciated and as is well known in the art, these experimentscan be massive in scale. They involve a huge labor force ranging fromscientists to field staff to design, plant, maintain, and conduct theexperiments, which can involve thousands or tens of thousands ofindividual plants. They also require substantial land resources. Plotsor greenhouses can take up thousands of acres of land. Not only doesthis tie up large amounts of land for months while the plants germinate,grow, and produce seed, during which time they may be tested in thelaboratory or field, but then the massive amounts of seed must beindividually tagged, harvested and processed.

A further complication is that much of the experimentation goes fornaught. It has been reported in the literature that some seed companiesdiscard 80-90% of the plants early on in the experiment. Thus, much ofthe land, labor and material resources expended for growing, harvesting,and post-harvest processing ultimately are wasted for a large percentageof the seed.

Timing pressures are also a factor. Significant advances in plantbreeding have put pressure on seed companies to quickly advance lines orvarieties of plants that have more and better traits andcharacteristics. The plant breeders and associated workers are thusunder increasing pressure to more efficiently and effectively processthese generations and make significant selections early on in thebreeding process.

Therefore, a movement towards earlier identification of traits ofinterest through laboratory based seed testing has emerged. Seed isnon-destructively tested to derive genetic, biochemical or phenotypicinformation. If traits of interest are identified, the selected seedfrom specific plants are used either for further experiments andadvancement, or to produce commercial quantities. Testing seed preventsthe need to grow the seed into immature plants, which are then tested.This saves time, space, and effort. If effective, early identificationof desirable traits in seed can lead to a great reduction in the amountof land needed for experimental testing, the amount of seed that must betested, and the amount of time needed to derive the informationnecessary for making advancement decisions. For example, instead ofthousands of acres of plantings and the subsequent handling andprocessing of all those plants, a fraction of acres and plants might beenough. However, because timing is still important, this is still asubstantial task because even such a reduction involves processing, forexample, thousands of seed per day.

A conventional method of attempting non-lethal seed testing is asfollows: a single seed of interest is held with pliers above a sheet ofpaper laid out on a surface; a small drill bit is used to drill into asmall location on the seed; debris is removed by the drill bit andcollected on a sheet of paper; the paper is lifted; and the debris istransferred to a test tube or other container for subsequent laboratoryanalysis. This method is intended to be non-lethal to the seed. However,the process is slow, and its success and effectiveness depends heavilyon the attention and accuracy of the worker. Each single seed must bemanually picked up and held by the pliers. The drilling is also manual.Care must be taken with the drilling and the handling of the debris.Single containers, e.g. the individual test tubes, must then be handledand marked or otherwise tracked and identified. Additionally, the pliersand drill must be cleaned between the testing of each seed. There can besubstantial risk of contamination by carry-over from seed to seed andthe manual handling. Also, many times it is desirable to obtain seedmaterial from a certain physiological tissue of the seed. For example,with corn seed, it may be desirable to genotype the endosperm. In suchcases, it is not trivial, but rather is time-consuming and somewhatdifficult, to manually grasp a small corn seed in such a way to allowthe endosperm to be oriented to expose it for drilling. Testing otherseed structures such as the seed germ is preferably avoided becauseremoving material from such regions of the seed negatively impactsgermination rates. Sometimes it is difficult to obtain a useful amountof material with this method.

Another example of non-lethally obtaining tissue from corn seed forlaboratory analysis is disclosed at V. Sangtong, E. C. Mottel, M. J.Long, M. Lee, and M. P. Scott, Serial Extraction of Endosperm Drillings(SEED) —A Method for Detecting Transgenes and Proteins in Single ViableMaize Kernels, Plant Molecular Biology Reporter 19: 151-158, June 2001,which is incorporated by reference herein. It describes use of ahand-held rotary grinder to grind off particles, called “drillings,”from the kernel and collection of the particles to test for the presenceof certain genes. However, this method also requires manual grasping andorientation of each individual seed relative to the grinder. It, too istime consuming and somewhat cumbersome. It also relies on the skill ofthe worker. This method raises issues of throughput, accuracy, whether auseful amount of material is obtained, and contamination. The grindermust be thoroughly cleaned between kernels in order to preventcontamination.

As evidenced by these examples, present conventional seed analysismethods used in genetic, biochemical, or phenotypic analysis, require atleast a part of the seed to be removed and processed. In removing someseed tissue, various objectives may need to be met. These may includeone or more of the following objectives:

(a) maintain seed viability after collection of seed tissue, ifrequired.

(b) obtain at least a minimum required amount of tissue, withoutaffecting viability.

(c) obtain tissue from a specific location on the seed, often requiringthe ability to orient the seed in a specific position.

(d) maintain a particular throughput level for efficiency purposes.

(e) reduce or virtually eliminate contamination.

(f) allow for the tracking of separate tissues and their correlation toseeds from which the tissues were obtained.

(a) Viability

With regard to maintaining seed viability, it may be critical in somecircumstances that the seed tissue removal method and apparatus notdamage the seed in such a way that seed viability is reduced. It isoften desirable that such analysis be non-lethal to the seed, or atleast result in a substantial probability that the seed will germinate(e.g. no significant decrease in germination potential) so that it canbe grown into a mature plant. For some analyses, seed viability does notneed to be maintained, in which case larger quantities of tissue canoften be taken. The need for seed viability will depend on the intendeduse of the seeds.

(b) Tissue Quantity

It is desirable to obtain a useful amount of tissue. To be useful, itmust be above a certain minimum amount necessary in order to perform agiven test and obtain a meaningful result. Different tests or assaysrequire different quantities of tissue. It may be equally important toavoid taking too much tissue to avoid reducing germination potential ofa seed, which may be undesirable. Therefore, it is desirable that theapparatus and methods for removing the seed tissue allow for variationin the amount of tissue taken from any given seed.

(c) Tissue Location

A useful amount of tissue also can involve tissue location accuracy. Forexample, in some applications the tissue must come only from a certainseed location or from specific tissue. Further, it is difficult tohandle small particles like many seeds. It is also difficult toaccurately position and orient seed. On a corn seed, for example, it maybe important to test the endosperm tissue, and orient the corn seed foroptimal removal of the endosperm tissue. Therefore, it is desirable thatthe apparatus and methods for removing the seed tissue are adapted toallow for location-specific removal, which may include specific seedorientation methods.

(d) Throughput

An apparatus and methodology for seed tissue removal must consider thethroughput level that supports the required number of tissues to betaken in a time efficient manner. For example, some situations involvethe potential need to test thousands, hundreds of thousands, or evenmillions of seed per year. Taking the hypothetical example of a millionseed per year, and a 5-day work week, this would average nearly fourthousand tests per day for each working day of a year. It is difficultto meet such demand with lower throughput methods. Accordingly, higherthroughput, automatic or even semi-automatic methods for removal of seedtissue may be desirable.

(e) Avoiding Contamination

It is desirable that an apparatus and methodology for seed tissueremoval not be prone to cross-contamination in order to maintain purityfor subsequent analytical testing procedures. This can involve not onlytissue location accuracy, such that tissue from a given location is notcontaminated with tissue from a different location, but also methodsinvolved in the removal and handling of the tissue to be tested,ensuring no contamination.

(f) Tracking Tissue to be Tested

Efficient processing of seeds and tissue removed from seeds presents avariety of challenges, especially when it is important to keep track ofeach seed, the tissue removed from such, and their correlation to eachother, or to other tissues. Accordingly, it is desirable that apparatusand methods for tissue removal and testing allow for easy tracking ofseed and tissue removed from such.

Conventional seed testing technologies do not address these requirementssufficiently, resulting in pressures on capital and labor resources, andthus illustrate the need for an improvement in the state of the art. Thecurrent methods are relatively low throughput, have substantial risk ofcross-contamination, and tend to be inconsistent because of a relianceon significant manual handling, orienting, and removal of the tissuefrom the seed. This can affect the type of tissue taken from the seedand the likelihood that the seed will germinate. There is a need toeliminate the resources current methods require for cleaning betweenremoval of individual portions of seed tissue. There is a need to reduceor minimize cross-contamination between unique tissue portions to betested by carry-over or other reasons, or any contamination from anysource of any other tissue. There is also a need for more reliabilityand accuracy. Accordingly, there is a need for methodologies and theircorresponding apparatus which provide for seed tissue removal andtesting that accomplishes one or more of the following objectives:

(a) maintains seed viability after seed tissue removal.

(b) obtains at least a minimum required amount of tissue, withoutaffecting viability.

(c) obtains tissue from a specific location on the seed.

(d) maintains a particular throughput level for efficiency purposes.

(e) reduces or virtually eliminate contamination.

(f) allows for the tracking of separate tissues and their correlation toseeds from which the tissues were obtained.

Some of these objectives can be conflicting and even antagonistic. Forexample, obtaining a useful amount of tissue while maintaining seedviability requires taking some seed tissue, but not too much. Moreover,high-throughput methodologies involve rapid operations but may beaccompanied by decreases in accuracy and increased risk ofcontamination, such that the methods must be done more slowly than istechnically possible in order to overcome the limitations. Thesemultiple objectives have therefore existed in the art and have not beensatisfactorily addressed or balanced by the currently available methodsand apparatuses. There is a need in the art to overcome theabove-described types of problems such that the maximum number ofobjectives is realized in any given embodiment.

SUMMARY

The invention includes methods for analyzing plant material, andspecifically seed tissue, while preserving viability of the seed orembryo (i.e. can form a plant). The method may include the steps ofcollecting shed cellular material from one or more embryos; obtaininggenetic material such as DNA from the shed cellular material; performinga molecular analysis of the genetic material; and germinating at leastone of said one or more embryos. The one or more embryos may beimmature. In one embodiment, the shed cellular material is collectedfrom an embryo by agitating the embryo in a non-destructive medium suchas water or other aqueous solution. In some embodiments, DNA may beobtained from the shed cellular material by exposing the shed cellularmaterial to cold and then heat followed by agitation; the steps may berepeated. In other embodiments, DNA may be obtained from the shedcellular material by heating of the shed cellular material andagitation; the steps may be repeated. In other embodiments, DNA may beobtained by incubating the shed cellular material with an enzyme; theenzyme may be VISCOZYME® L, a multi-enzyme complex containing a widerange of carbohydrases, including arabanase, cellulase, β-glucanase,hemicellulase, and xylanase. (See the Sigma Aldrich product catalog). Instill other embodiments, DNA may be obtained using DNA extractiontechniques, such as but not limited to the use of magnetic particlesthat bind genetic material or any method known to one of ordinary skillin the art.

The methods of the invention include obtaining genetic material fromembryos and performing a molecular analysis of the genetic materialwhile preserving the embryos' ability to germinate. In some embodiments,the embryos are suspended in an aqueous solution surrounded by a matrixof one or more oils. Preferably, at least one of the one or more oilshas a density greater than that of the aqueous solution. The one or moreembryos may be immature. In some embodiments, antimicrobial agentsand/or minimal growth media may be added to the aqueous solution. Insome embodiments, the embryos may be stored in cold and/or darkconditions to prevent premature germination. In a preferred embodiment,the embryos are stored at a temperature of approximately 4° C. In someembodiments, the embryos may be transferred for continued storage. Inother embodiments, the embryos may be transferred to germination medium,and one or more of the embryos may be germinated. In still otherembodiments, an aliquot of the aqueous solution may be removed, geneticmaterial may be obtained from cellular material in the aliquot, and thegenetic material may be used for molecular analysis (e.g. to genotypethe stored embryos). The molecular analysis may be genotyping, which mayoccur by way of: single nucleotide polymorphism detection, restrictionfragment length polymorphism identification, random amplifiedpolymorphic detection, amplified fragment length polymorphism detection,polymerase chain reaction, DNA sequencing, whole genome sequencing,allele specific oligonucleotide probes, or DNA hybridization to DNAmicroarrays or beads. Whole genome amplification may be performed priorto the molecular analysis. In other embodiments, one or more of thesteps described above may be automated.

Methods of the invention include obtaining embryonic DNA (whether or notsaid obtaining the embryonic DNA includes extraction), storing theembryo from which the DNA was extracted in a manner that preserves theembryo's ability to germinate and grow into a plant, genotyping theembryo using the embryonic DNA, and determining whether to germinate andgrow the embryo (i.e. selecting) or to discard the embryo based on itsgenotype (i.e. counterselecting). An embryo that is selected togerminate and grow based on its genotype may be grown into a plant andphenotyped, used for breeding, or used to bulk up seed of the samegenotype. In preferred embodiments, one or more steps of the method maybe automated.

One embodiment of the invention allows for determining the maternallineage of one or more seeds by collecting maternal seed tissue from theone or more seeds; washing the maternal seed tissue; dissociating andhomogenizing the maternal seed tissue to obtain a homogenized solution;centrifuging the homogenized solution to obtain supernatant; andperforming a molecular analysis using supernatant DNA. In oneembodiment, the maternal seed tissue is pericarp. The washing step maybe performed with 1% sodium dodecyl sulfate solution, water, ethanol, ormixtures thereof. The washing step is preferably performed with anaqueous solution of about 1% sodium dodecyl sulfate. The dissociatingand breaking pericarp tissue may be performed using a cell dissociator(such as gentleMACS™, Miltenyi Biotech). The method may further compriseusing whole genome amplification prior to the molecular analysis toobtain sufficient DNA yield.

Another embodiment of the invention allows for determining the maternallineage of one or more seeds by collecting maternal seed tissue from theone or more seeds; washing the maternal seed tissue; dissociating andhomogenizing the maternal seed tissue to obtain a homogenized solution;extracting DNA from cells contained within the homogenized solution; andperforming a molecular analysis of the extracted DNA. In one embodiment,the maternal seed tissue is pericarp. The washing step may be performedwith 1% sodium dodecyl sulfate solution, water, ethanol, or mixturesthereof. The washing step is preferably performed with an aqueoussolution of about 1% sodium dodecyl sulfate. The dissociating andhomogenizing step may be performed using a cell dissociator (such asgentleMACS™, Miltenyi Biotech). The extracting step may be performedusing DNA-binding magnetic particles or Extract-N-Amp™. The method mayfurther comprise using whole genome amplification prior to the molecularanalysis to obtain sufficient DNA yield.

Another embodiment of the invention allows for determining the maternallineage of one or more seeds by collecting maternal seed tissue from theone or more seeds; washing the maternal seed tissue; disrupting thematernal seed tissue in liquid nitrogen; extracting DNA from thedisrupted maternal seed tissue; and performing a molecular analysis ofthe extracted DNA. In one embodiment, the maternal seed tissue ispericarp. The washing step may be performed with 1% sodium dodecylsulfate solution, water, ethanol, or mixtures thereof. The washing stepis preferably performed with an aqueous solution of about 1% sodiumdodecyl sulfate. The extracting step may be performed using DNA-bindingmagnetic particles or Extract-N-Amp™. The method may further compriseusing whole genome amplification prior to the molecular analysis toobtain sufficient DNA yield.

Another embodiment of the invention allows for determining the maternallineage of one or more seeds by collecting maternal seed tissue from theone or more seeds; washing the maternal seed tissue; extracting DNAdirectly from the washed maternal seed tissue; and performing amolecular analysis of the extracted DNA. In one embodiment, the maternalseed tissue is pericarp. The washing step may be performed with 1%sodium dodecyl sulfate solution, water, ethanol, or mixtures thereof.The washing step is preferably performed with an aqueous solution ofabout 1% sodium dodecyl sulfate. The extracting step may be performedusing Extract-N-Amp™. The method may further comprise using whole genomeamplification prior to the molecular analysis to obtain sufficient DNAyield.

In any of the embodiments stated above, the molecular analysis may begenotyping. When maternal seed tissue from more than one seed replicateis collected, a consensus genotype may be obtained.

DESCRIPTIONS OF THE DRAWINGS

In FIGS. 1 through 11, upside down triangles represent samples havingone homozygous state; right side up triangles represent samples havingthe other homozygous state; triangles pointing towards the leftrepresent the heterozygous control; circles represent missing ornegative control data; and diamonds represent unquantifiable calls. Thetighter the cluster of points along a line parallel to either axis, theless variation with the method being tested.

FIG. 1 depicts genotyping data from one marker using DNA obtained withthe cold-heat shock treatment. The data represents three differenttreatments (incubate only; incubate and tap; and incubate, tap, andspin) in each of four different incubation volumes (10 μL, 20 μL, 50 μL,and 75 μL).

FIG. 2 depicts genotyping data from one marker using DNA obtained withthe cold-heat shock treatment. The data represents one treatment(incubate, tap, and spin) in an incubation volume of 50 μL.

FIG. 3 depicts genotyping data from one marker using DNA obtained fromcold-heat shock, heat shock, incubation with VISCOZYME® L, or DNAextraction using the Sbeadex method. The data represents three differenttreatments (incubate only; incubate and tap; and incubate, tap, andspin) in an incubation volume of 50 μL.

FIG. 4 depicts genotyping data from one marker using DNA obtained withthe cold-heat shock treatment. The data represents one treatment(incubate, tap, and spin) in an incubation volume of 50 μL.

FIG. 5 depicts genotyping data from one marker using DNA obtained fromcold-heat shock, incubation with VISCOZYME® L, or DNA extraction usingthe Sbeadex method. The data represents three different treatments(incubate only; incubate and tap; and incubate, tap, and spin) in anincubation volume of 50 μL.

FIG. 6 depicts genotyping data from one marker using DNA obtained withthe cold-heat shock treatment. The data represents one treatment(incubate, tap, and spin) in an incubation volume of 50 μL.

FIG. 7 depicts genotyping data from one marker using DNA obtained withthe cold-heat shock treatment. The data represents three treatments(incubate only; incubate and tap; and incubate, tap, and spin) in anincubation volume of 150 μL.

FIG. 8 depicts genotyping data from one marker using DNA obtained withthe cold-heat shock treatment. The data represents one treatment(incubate, tap, and spin) in an incubation volume of 150 μL.

FIG. 9 depicts genotyping data from one marker using DNA obtained withthe cold-heat shock treatment or no treatment at all following washingof the shed cellular material. The data represents three treatments(incubate only; incubate and tap; and incubate, tap, and spin) and twoincubation volumes (50 μL and 100 μL).

FIG. 10 depicts genotyping data from one marker using DNA obtained withthe cold-heat shock treatment. The data represents one treatment(incubate, tap, and spin) in an incubation volume of 50 μL.

FIG. 11 depicts genotyping data from one marker using DNA obtained withthe cold-heat shock treatment and whole genome amplification (using theREPLI-g Single Cell Kit) to obtain sufficient yield of DNA prior togenotyping. The data represents four treatments (incubate only; vortexat speed 3 for 5 seconds; vortex at speed 10 for 5 seconds; and vortexat speed 10 for 30 seconds) in an incubation volume of 10 μL.

FIG. 12 depicts germination results for embryos of a first maize line,wherein the embryos were stored using methods of the invention.

FIG. 13 depicts germination results for embryos of a second maize line,wherein the embryos were stored using methods of the invention.

FIGS. 14a-14c depict the steps involved in peeling of pericarp tissue.The back side of the kernel (farthest from the embryo) may be cut with asharp blade, as shown in FIG. 14a . Preferably, the blade is sterilizedafter the first cut before outer edge of the kernel may be cut with thesharp blade, starting from one end of the first cut, around the edge ofthe kernel, and down to the other end of the first cut, as shown in FIG.14b . Sterilized forceps may be used to peel the pericarp tissue fromthe kernel as shown in FIG. 14 c.

FIG. 15 compares the ILLUMINA® GOLDENGATE® Genotyping Assay using DNAobtained from a) conventional CTAB DNA extraction method using multipleseeds and b) SBEADEX® DNA extraction method using one seed (with tissuewash) followed by the whole genome amplification.

FIG. 16 demonstrates that quality fluorescent marker data can beobtained from a single pericarp.

FIG. 17 demonstrates the high degree of similarity between the measuredgenotype of the pericarp tissue extracted from a single seed (each line)and the known maternal genotype.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Genotyping of embryos or other seed parts permits molecularcharacterization early in plant development, allowing selections of adesired genotype to be made weeks or months earlier than other methodssuch as with phenotyping or plant genotyping. Consequently, resourcescan be focused earlier on embryos that have the highest probability ofdeveloping into desirable plants. Techniques for geneticallycharacterizing seed tissue can greatly enhance a molecular breedingprogram and eliminate a great deal of effort and resources by allowingbreeders to only grow plants with the desired genetics. Furthermore, theability to reliably genetically characterize an embryo without impedingits ability to germinate, particularly in an immature embryo, cansubstantially reduce the amount of time required between generations ofplants.

Non-destructive genotyping in a plant breeding program may require oneor more of the following steps:

-   -   1. Separating viable plant sources from other plant material;    -   2. Preserving the viable plant sources;    -   3. Obtaining genetic material corresponding to multiple viable        plant sources while maintaining the viability of the multiple        viable plant sources;    -   4. Obtaining genetic material for molecular characterization;    -   5. Molecularly characterizing the genetic material from the        multiple viable plant sources;    -   6. Selecting one or more viable plant sources based on molecular        characterizations; and    -   7. Growing the selected viable plant sources.

The viable plant sources may be seeds, plant embryos, plant tissue, orwhole plants, for example. Most typically, viable plant sources arecapable of being grown into plants, although not necessarily. Thegenetic material may be crude, i.e., mixed with other portions of planttissue including cellulosic and protein materials, or it may be purified(such as, for example, by DNA extraction methods known to one ofordinary skill in the art). The genetic material may be taken directlyfrom the viable plant sources, or it may be taken from other plantmaterial. The preserving step may include keeping the viable plantsources in a manner that preserves an ability to be grown into a plant.The preserving step may include keeping the viable plant sources in amanner that prevents germination. The molecularly characterizing stepmay involve genotyping, genetic sequencing, RNA sequencing, restrictionfragment length polymorphism marker detection, single nucleotidepolymorphism detection, whole genome amplification, specific proteindetection, oil content measurement, protein content measurement, or anyother molecular analysis that may serve as a basis to select or rejectparticular viable plant sources. The growing step may involve any meansof growing plants, including planting in a field or a greenhouse,growing hydroponically, growing aeroponically, or any other method ofgrowing a plant. In some embodiments, the plant is grown to maturity andproduces pollen and/or seeds. In some embodiments, one or more of thesteps is automated.

Separating Viable Plant Sources.

In one embodiment involving corn, the caps of corn kernels are slicedoff while they are still attached to the corn cob. The caps of the cornkernels are typically the farthest part of the kernel from the embryo,which is closer to the tip of the kernel, which is attached to the cob.Each embryo may then be removed, for example, using a small spatula orany other suitable device. In one embodiment, this process is automatedusing a robot cap slicer, a robotically manipulated spatula, and amachine vision platform for precise cutting and embryo removal control.

In another embodiment, corn kernels may be removed from the cob beforeembryo removal. The kernels may then be oriented in the same way, forexample, by floating the kernels in water or in a solution. The kernelsmay then be immobilized, while preserving their orientations, forexample, by draining them into a container with multiple wells, eachwell holding an oriented kernel. Small pieces of the tips of the kernelsmay then be removed, preferably small enough pieces of the tip of thekernels are removed to avoid damaging the embryos. The embryos may thenbe extracted by gently squeezing the kernels from the cap sides of thekernels.

Following embryo removal, each embryo may be placed in a container withmultiple wells, wherein the location of each embryo in each well isrecorded, associated, or correlated with the location of geneticmaterial obtained in a subsequent step.

Preserving the Viable Plant Sources

When the viable plant sources are seeds, preservation of seeds for thequantity of time required to perform a molecular analysis typicallyrequires no particular care. When the viable plant sources are embryos,however, special care should be taken to preserve viability. Embryos maybe stored in a multiple well plate, where each well corresponds to awell in which extracted tissue to be tested is placed.

In one preferred method, embryos are suspended in an aqueous solutionsurrounded by a matrix of one or more oils. An oil having a density lessthan water will cover the embryo(s) in the aqueous solution, while anoil having a density greater than water will support the embryo(s) inthe aqueous solution. In some embodiments, the one or more embryos issuspended in an aqueous solution surrounded by a matrix of two or moreoils, wherein at least one of the two or more oils is more dense thanthe aqueous solution and at least one of the two or more oils is lessdense than the aqueous solution, further wherein the aqueous solution issurrounded by the oil that is more dense than the aqueous solution andthe oil that is less dense than the aqueous solution. In someembodiments, antimicrobial agents and/or minimal growth media may beadded to the aqueous solution. In some embodiments, the embryos may bestored in cold and/or dark conditions to prevent premature germination.In a preferred embodiment, the embryos are stored at a temperature ofapproximately 4° C. In some embodiments, the embryos may be transferredfor continued storage. In other embodiments, the embryos may betransferred to germination medium, and the embryos may be germinated. Ina preferred embodiment, an aliquot of the aqueous solution may beremoved; genetic material may be obtained from cellular material in thealiquot; and the genetic material may be used for molecular analysis(e.g. to genotype the stored embryos).

High density oil that may be used in this method includes but is notlimited to perfluoro compounds having 12 compounds (e.g., DuPont's lowerviscosity KRYTOX® oils). Low density oil that may be used in this methodincludes but is not limited to phenylmethylpolysiloxane. Other non-toxicoils known to those of ordinary skill in the art may be used instead ofor in combination with these compounds.

Obtaining Genetic Material.

The invention includes many different options for the step of obtaininggenetic material (e.g. DNA). Genetic material may be obtained from any“shed cellular material”, which refers to any plant material remainingafter the separation of viable plant sources. Shed cellular material mayinclude embryo and/or endosperm material. If genetic information for theparent plant is desired, genetic material may be obtained from thepericarp.

In one embodiment, a small piece of the scutellum may be excised usingany method known in the art, include cutting with a blade or a laser.Preferably, the piece of the scutellum is small enough so as not tocompromise embryo viability. The embryo and corresponding piece ofscutellum may then be placed in separate containers with wells, in whichthe well containing the embryo in the embryo container and the wellcontaining the corresponding scutellum in the scutellum container arecorrelated such that any information gained from the scutellum isassociated with the embryo from which the scutellum tissue was obtained.

In another embodiment, when a spatula (or any other implement or deviceused to excise a piece of the scutellum) is used to remove the embryofrom a seed, the spatula may then be dipped into a well in one containerthat corresponds to a well in a second container that houses the embryo.Preferably, the spatula is dipped into a well containing an aqueoussolution. When the spatula is used to remove the embryo, sufficientquantities of endosperm tissue remain on the spatula (i.e. shed cellularmaterial), and the spatula need not contact the kernel from which theembryo was removed following embryo extraction. The spatula may bedipped in the well containing aqueous solution immediately after theembryo has been removed. If the same spatula is used for the removal ofmultiple embryos and/or endosperm tissue, it preferably will be cleanedbetween each use to remove any genetic material that could lead tocontamination.

In another embodiment, the embryo may be washed, for example with water,to remove any endosperm attached to the embryo. The washed embryo maythen be immersed in fresh water or other aqueous solution and agitatedto remove a small number of embryo cells from the embryo into the freshwater or other aqueous solution (i.e. shed cellular material). Theembryo may then be transferred to a container with multiple wells, andsome or all of the fresh water or aqueous solution containing the smallnumber of embryo cells may be transferred to a correlated well in aseparate container with multiple wells.

In another embodiment not necessarily requiring embryo extraction orother separation of viable plant sources, a piece of the outer coat of acorn kernel, the pericarp, may be excised in order to conduct amolecular analysis of the parent plant. In this embodiment, kernels maybe soaked in water before making cuts in the pericarp. The back side ofthe kernel (farthest from the embryo) may be cut with a sharp blade, asshown in FIG. 14a . Preferably, the blade is sterilized after the firstcut before outer edge of the kernel may be cut with the sharp blade,starting from one end of the first cut, around the edge of the kernel,and down to the other end of the first cut, as shown in FIG. 14b .Sterilized forceps may be used to peel the pericarp tissue from thekernel as shown in FIG. 14c . While the cut can be made on the frontside of the kernel (nearest the embryo), the cut is preferably made onthe back side to reduce the possibility that the pericarp will becontaminated with endosperm tissue. To further reduce the possibility ofcontamination, the pericarp tissue may be washed after it is excised.The pericarp may be placed in the well of a container and the seed fromwhich the pericarp was excised (or the embryo from that seed) may beplaced in a corresponding well of a separate container. As will beunderstood by those of ordinary skill in the art, there are othercomparable methods for isolating pericarp tissue, and in someembodiments of the invention, pericarp DNA may be extracted withoutpericarp removal.

The tissue to be analyzed is preferably associated or correlated withits corresponding viable plant source so that the corresponding viableplant source can be selected based on the results of the tissueanalysis.

Obtaining Genetic Material for Molecular Characterization

In order for genetic material to be analyzed, it must be freed from thecell such that it is accessible for molecular analysis. This may involvephysical treatments such as exposure to cold-heat or just heat,incubation with enzymes, or even DNA extraction techniques (although itis important to note that extraction is not a necessary step inobtaining DNA for molecular analysis). Essentially any process thatdisrupts the tissue and breaks open cells, thereby releasing DNA thatcan be used for molecular characterization, may be used in the methodsprovided herein.

In some embodiments, DNA may be obtained from the shed cellular materialby exposing the shed cellular material to cold-heat or heat, agitatingthe mixture, and optionally repeating. In other embodiments, DNA may beobtained by incubating shed cellular material with an enzyme; the enzymemay be VISCOZYME® L, a multi-enzyme complex containing a wide range ofcarbohydrases, including arabanase, cellulase, R-glucanase,hemicellulase, and xylanase. (See the Sigma Aldrich product catalog). Instill other embodiments, obtaining DNA may comprise extraction of theDNA, such as through the use of magnetic particles that bind geneticmaterial or any method known to one of ordinary skill in the art.However, extraction is not necessary for obtaining DNA.

In other embodiments involving maternal seed tissue such as pericarptissue, tissue may be dissociated using a cell dissociator (such asgentleMACS™, Miltenyi Biotech), optionally followed by DNA extraction.In another embodiment, the maternal seed tissue may be disrupted inliquid nitrogen prior to DNA extraction. In yet another embodiment, DNAmay be extracted directly from washed maternal seed tissue (e.g. usingExtract-N-Amp™).

Molecularly Characterizing the Genetic Material from the Multiple ViablePlant Sources

In cases where the yield of DNA obtained from embryo tissue is notsufficient for some molecular analysis (e.g. high density genotyping),whole genome amplification techniques may be used. The Qiagen REPLI-gkit, the Sigma-Aldrich SeqPlex kit, or any other technique known to oneof ordinary skill in the art may be used to amplify DNA from embryotissue.

Other useful molecular characterizations may involve sequencing all orpart of the genome of the tissue extracted from the seed, or usingmolecular markers and fluorescent probes to genotype. Molecularcharacterization need not focus on the genotype of the extracted tissue,but instead may measure other properties such as oil content, oilcomposition, protein content, or the presence or absence of particularmolecules in the tissue.

In a preferred embodiment, genetic material is placed in a well of amultiple well plate containing a bilayer of oil, one layer having adensity greater than water and one layer having a density less thanwater. Multiple wells contain multiple different genetic materials.Fluorescently labeled probes are added to the genetic materials, andthermocycling to cause amplification and hybridization of the probes isperformed in the multiple well plate. The wells are irradiated andfluorescence is detected from the labels to generate genotypic data.Alternatively, the genetic material may be sequenced, in whole or inpart, in the multiple well plate.

Selecting One or More Viable Plant Sources Based on MolecularCharacterizations

In a molecular breeding program, plants or potential plants are selectedto participate in subsequent generations based on their genotype.Typically this involves determining whether the plant has inherited oneor more desirable traits indicated by genetic markers whose presence orabsence can be determined based on the genotyping. Plant breeders selectthose plants that have the desired traits to participate in furtherbreeding, to inbreed, or as part of a process to create inbreds throughhaploid doubling techniques.

Growing the Selected Viable Plant Sources.

Those plants that are selected based on the presence of desirable traitsas determined by their genotype may be grown into mature plants, toobtain haploid material to create a double haploid inbred, to breed withitself to create an inbred, or to breed with other plants to improve anddiversify germplasm.

In one embodiment, a consensus genotype may be derived by consideringgenotypic data from multiple tissue specimens obtained from one or moreseeds, each tissue specimen being a replicate. In a genotypingexperiment that identifies multiple nucleotides across multiplepositions in a genome, it is not uncommon for any particular experimentto fail to identify one or more of the nucleotides to be identified.Thus, missing nucleotide identifications for each missing position maybe noted for each of the specimens. If a nucleotide identification fromonly one specimen is available for a particular nucleotide position,then that nucleotide identification is assigned as the consensus datafor that position. If two or more nucleotide identifications areavailable for a particular nucleotide position, then the majority ofnucleotide identifications for that position is assigned as theconsensus data for that position. If no majority identification existsfor a position, that position is assigned as missing data for theconsensus genotype. The probabilities for consensus accuracy for a givennucleotide position is given in Table 1 for the cases of 1, 2, 3, and 4replicates, where f represents the error rate in genotyping (e.g.,marker call).

TABLE 1 Probabilities of Consensus Accuracy Available Same CallReplicates (Consensus) Different Call Probability 1 1 0 1-f  2 2 0 1-f²2 1 1 0.5 3 3 0 1-f³ 3 2 1 1-3*(1-f)*f² 4 4 0 1-f⁴ 4 3 1 1-4*(1-f)*f³ 42 2 0.5

While the examples provided here relate to obtaining and genotypingtissues from a monocot, specifically maize, those of ordinary skill inthe art would understand how to apply the same or similar methods toother monocots and dicots; the methods may be adapted to any plant.Further, the genotyping methods disclosed herein may be used to genotypeany plant tissue. The consensus genotyping methods may also be used togenerate a consensus genotype for multiple specimens of any geneticmaterial obtained from any source without departing from the stepsdisclosed.

Example 1 Embryo Genotyping

A. Collection of Embryo Material:

Embryos were washed 3 times using 2 mL of sterile water. Embryos wereincubated in a tube containing 10 μL, 20 μL, 50 μL 75 μL, or 150 μL ofsterile water for either 10 minutes, 20 minutes, or overnight. It wasfound that adequate genotyping data can be obtained with any of thedilution volumes, and that 10 minutes was a sufficient incubation time.All protocols for washing and incubating the embryos were used with allthree tissue collection methods described below.

Method 1: The tubes containing the embryos were agitated via tapping 10times and were then spun down in a tabletop centrifuge for 5 seconds.The water was then removed from each tube for analysis. It was foundthat this method achieved the best results for genotyping.

Method 2: Embryos were washed 3 times using 2 mL of sterile water.Embryos were incubated in a tube containing 50 μL of sterile water for10 minutes. The water was then removed from the tube for analysis.

Method 3: Embryos were washed 3 times using 2 mL of sterile water.Embryos were incubated in a tube containing 50 μL of sterile water for10 minutes. Tubes containing the embryos were agitated via tapping 10times. The water was then removed from each tube for analysis.

B. Methods to Obtain DNA:

Cold-Heat Shock:

Embryo material obtained using all three methods described above wasplaced in a −80° C. freezer for 20 min; then placed on a thermocycler at100° C. for 10 min and pipetted up and down to mix. The process wasrepeated for a total of two rounds. The resulting mixtures were storedat −20° C. It was found that the best results for genotyping wereachieved from DNA obtained using this method.

Heat Shock Only:

Embryo tissues were placed on a thermocycler at 100° C. for 10 min andpipetted up and down to mix. The process was repeated for a total of tworounds. The mixtures were stored at −20° C.

Enzymatic Method:

The mixtures from the preceding step were incubated in a 95° C. oven toevaporate off the remaining water. 18.0 μL of PBS solution and 2.0 μL ofdiluted VISCOZYME® L (commercially available from Sigma-Aldrich; diluted1:200 in PBS Solution pH 7.4; total vol. 20 μL) were added and themixtures were incubate at 37° C. for 2 hours. A quantity of 2.0 μL ofdiluted proteinase K (commercially available from Sigma-Aldrich; diluted1:20 in PBS Solution pH 7.4) was added and the mixtures were incubatedat 55° C. for 50 minutes then heated to 95° C. for 10 min. The mixtureswere stored at −20° C.

DNA Extraction:

The mixtures from the methods of Example 1 B were incubated in a 95° C.oven to evaporate off the remaining water. 45 μL Lysis buffer PN (LGCGenomics) was added to each mixture, which were then centrifuged brieflyand incubated at 65° C. for 1 hour. To new tubes were added 60 μLBinding buffer PN, 5 μL Sbeadex particles (magnetic particles that bindgenetic material, which are commercially available from LGC Genomics)followed by the lysate mixtures, which were then incubated at roomtemperature for 4 minutes to allow binding of DNA to the particles,vortexed briefly and placed in a magnetic rack to concentrate beads. Thelysis buffer was removed and 100 μL wash buffer PN1 (LGC Genomics) wereadded to resuspend the beads. Washing was repeated using 100 μL washbuffer PN2 (LGC Genomics) followed by a 100 μL pure water wash. 10 μLelution buffer PN was added and the mixtures were incubated at 55° C.for 10 minutes with vortexing every 3 minutes. The magnetic rack wasused to concentrate beads and the eluate was transferred to new tubesand stored at −20° C.

C. Whole Genome Amplification

When whole genome amplification was required the following protocol wasfollowed using the REPLI-g® Single Cell Kit (commercially available fromQiagen). Whole genome amplification was done to achieve higher DNA yieldand to facilitate the detection of high density marker sets.

2.5 μL template DNA was combined with 2.5 μL Buffer D1 (commerciallyavailable from Qiagen; total volume 5.0 μL) and incubated at roomtemperature for 3 minutes. 5.0 μL Buffer N1 (commercially available fromQiagen; total volume 10.0 μL) was added and the mixtures were vortexedand centrifuged briefly. A Master Mix containing 9.0 μL nuclease-freewater, 29.0 μL REPLI-g® Reaction Buffer (commercially available fromQiagen) and 2.0 μL REPLI-g® DNA Polymerase (commercially available fromQiagen) was used per reaction to give 50.0 μL total volume. The mixtureswere run on a thermocycler using a 30° C. for 8 hours and 4° C.thereafter. DNA quantitation was performed using a Qubit assay(commercially available from Life Technologies). The DNA product wasused directly in the genotyping step.

D. Molecular Analysis

TAQMAN® Marker Analysis

Marker analysis was carried out using TAQMAN® assays (commerciallyavailable from Life Technologies). DNA was diluted to a targetconcentration of 20 ng/μL. A 384 plate containing the DNA was loadedinto LC480 real-time PCR thermocycler and run using the followingprogram: pre-incubation: 1 cycle (95° C. for 5 minutes); amplification:45 cycles, (−95° C. for 30 seconds, −60° C. for 45 seconds (singleacquisition), −72° C. for 1 minute (single acquisition); cooling: 1cycle, (−72° C. for 10 minutes, −40° C. for 30 seconds). Calls were readusing Roche LC480 LightCycler® Software (commercially available fromRoche Diagnostics).

Results

The foregoing methods all gave acceptable genotyping results. Genotypicdata is shown in FIGS. 1-11, which include data from all permutations ofthe methods disclosed in this example. FIG. 1 depicts genotyping datafrom one marker using DNA obtained with the cold-heat shock treatment.The data represents three different treatments (incubate only; incubateand tap; and incubate, tap, and spin) in each of four differentincubation volumes (10 μL, 20 μL, 50 μL, and 75 μL). FIG. 2 depictsgenotyping data from one marker using DNA obtained with the cold-heatshock treatment. The data represents one treatment (incubate, tap, andspin) in an incubation volume of 50 μL. FIG. 3 depicts genotyping datafrom one marker using DNA obtained from cold-heat shock, heat shock,incubation with VISCOZYME® L, or DNA extraction using the Sbeadexmethod. The data represents three different treatments (incubate only;incubate and tap; and incubate, tap, and spin) in an incubation volumeof 50 μL. FIG. 4 depicts genotyping data from one marker using DNAobtained with the cold-heat shock treatment. The data represents onetreatment (incubate, tap, and spin) in an incubation volume of 50 μL.FIG. 5 depicts genotyping data from one marker using DNA obtained fromcold-heat shock, incubation with VISCOZYME® L, or DNA extraction usingthe Sbeadex method. The data represents three different treatments(incubate only; incubate and tap; and incubate, tap, and spin) in anincubation volume of 50 μL. FIG. 6 depicts genotyping data from onemarker using DNA obtained with the cold-heat shock treatment. The datarepresents one treatment (incubate, tap, and spin) in an incubationvolume of 50 μL. FIG. 7 depicts genotyping data from one marker usingDNA obtained with the cold-heat shock treatment. The data representsthree treatments (incubate only; incubate and tap; and incubate, tap,and spin) in an incubation volume of 150 μL. FIG. 8 depicts genotypingdata from one marker using DNA obtained with the cold-heat shocktreatment. The data represents one treatment (incubate, tap, and spin)in an incubation volume of 150 μL. One of the homozygous calls wasincorrect. FIG. 9 depicts genotyping data from one marker using DNAobtained with the cold-heat shock treatment or no treatment at allfollowing washing of the shed cellular material. The data representsthree treatments (incubate only; incubate and tap; and incubate, tap,and spin) and two incubation volumes (50 μL and 100 μL). FIG. 10 depictsgenotyping data from one marker using DNA obtained with the cold-heatshock treatment. The data represents one treatment (incubate, tap, andspin) in an incubation volume of 50 μL. FIG. 11 depicts genotyping datafrom one marker using DNA obtained with the cold-heat shock treatmentand whole genome amplification (using the REPLI-g Single Cell Kit) toobtain sufficient yield of DNA prior to genotyping. The data representsfour treatments (incubate only; vortex at speed 3 for 5 seconds; vortexat speed 10 for 5 seconds; and vortex at speed 10 for 30 seconds) in anincubation volume of 10 μL.

Example 2 Embryo Storage

Two lines of maize germplasm were selected for testing the impacts ofextended embryo storage in an oil matrix on germination rates. Embryosfrom each line were isolated by hand before being placed into theirrespective storage condition. All embryos were plated on germinationmedia to evaluate germination rates in a controlled growth chamber. Sixembryos of each line were immediately plated on germination mediawithout any storage exposure to act as a control for germination in acontrolled growth chamber. Seventy two (72) embryos of each line wereisolated and evenly divided across three storage conditions, with adedicated storage tube for each embryo:

Storage condition 1: 24 embryos were placed in 50 μL aqueous solutionsurrounded by two layers of oil with significantly different densities,one with a density significantly greater than water and one with adensity significantly less than water.

Storage condition 2: 24 embryos were placed in a 50 μL droplet ofaqueous solution with an added antimicrobial agent, surrounded by thetwo oils of condition 1.

Storage condition 3: 24 embryos were placed in a 50 μL droplet ofminimal growth media with an added antimicrobial agent, surrounded bythe two oils of condition 1.

All tubes were placed in a dark refrigerator at 4 degrees centigrade forthe duration of the experiment. At four (4) time points, 6 embryos ofeach line were removed from their storage condition and plated ongermination media in a controlled growth chamber to evaluate germinationrates. The time points were as follows:

Time point 1: 15 minutes after placement into storage.

Time point 2: 1 day after placement into storage.

Time point 3: 5 days after placement into storage.

Time point 4: 10 days after placement into storage.

Embryo germination rates were then monitored to determine optimalstorage conditions. The results of these tests are shown in FIGS. 12 and13 (results for two different lines of maize). It was found thatgermination rates were excellent in each of the three storage methods.

Example 3 Pericarp Genotyping

A. Pericarp Peeling

Kernels of corn were removed from the cob and soaked for 60 minutes indeionized water. A scalpel blade was sterilized using a bead sterilizer.The scalpel was used to cut the back side of the seeds (away from theembryo) near the tips, as shown in FIG. 14a . The scalpel was againsterilized using a bead sterilizer and cooled in sterile water. Thescalpel was then used to cut along the outer edge of the kernel, asshown in FIG. 14b . Forceps were sterilized in a bead sterilizer,cooled, then used to peel the pericarp from the kernel, as shown in FIG.14c . The pericarp tissue from each kernel was then placed inmicrocentrifuge tubes.

B. Pericarp Washing

Three different washing solutions were tested. The best results wereachieved washing with 1% sodium dodecyl sulfate (SDS) solution, althoughadequate results were achievable using water and ethanol. An alternativewashing method using sonication also gave adequate results. The washingprotocol used began by adding 1 mL wash solution to the microcentrifugetubes, which was placed in an inverter for 1 minute. The wash solutionwas removed and replaced with 1 mL fresh wash solution, then themicrocentrifuge tubes were again placed in an inverter, this time for 4minutes. The pericarp tissue was then removed, rinsed with distilledwater, and placed into a new microcentrifuge tube. The sonicationprotocol placed the pericarp tissue in a sonicator for 1 minute. Thetissue was then removed, rinsed with distilled water, and placed in afresh microcentrifuge tube.

C. Obtaining DNA

Five methods for obtaining DNA were tested. The best results wereachieved with the gentleMACS™ protocol with water or TE supernatants.

gentleMACS™/Water or TE Supernatants:

In this method, pericarp tissue was placed directly onto the rotor of agentleMACS™ M tube. 300 ul of water or TE buffer was added to the tube,which was then closed and placed in a gentleMACS™ machine. The automatedprogram “Protein_01.01” was run. For pericarp tissues that were notfully dissociated, further mixing and running of the automated programwas done. Next, the mixtures were spun down in the GentleMACS™ tube andtransferred to a new 1.5 ml Eppendorf tube. The Eppendorf tube was thencentrifuged at 14000 rpm for 2 minutes, and the supernatant weretransferred to a fresh 1.5 ml Eppendorf tube for the molecular analysis.No extraction of DNA was required in this method.

GentleMACS™/SBEADEX® in this Method, Pericarp Tissue was Placed Directlyonto the rotor of a gentleMACS™ M tube.

300 μL of SBEADEX® Lysis Buffer PN was added to the tube, which was thenclosed and placed in a gentleMACS™ machine. The automated program“Protein_01.01” was run. For pericarp tissues that were not fullydissociated, further mixing and running of the automated program wasdone. Next, the mixtures were centrifuged and incubated at 65° C. for 1hour with occasional agitation. 360 μL of Binding Buffer PN and 30 μLSBEADEX® particles were added to fresh 1.5 mL Eppendorf tubes. The tubeswith the pericarp tissue were centrifuged and the lysate was transferredto the fresh tubes. These were then incubated at room temperature for 4minutes to allow the DNA to bind to the SBEADEX® particles. The tubeswere then vortexed briefly then placed in a magnetic rack to concentratethe beads. The lysis buffer was removed and 600 μL of wash buffer PN1was added to each tube and the beads were resuspended. The tubes wereagain placed in a magnetic rack to concentrate the beads and the washbuffer PN1 was removed. This washing procedure was repeated using 600 μLof wash buffer PN2, then repeated again using 600 μL of pure water.Following this third washing step, 40 μL of elution buffer PN was addedand the tubes were incubated at 55° C. for 20 minutes and vortexed every3 minutes. A magnetic plate was used to concentrate the beads, and theeluate was transferred into fresh tubes, then stored at −20° C. untilmolecular characterization.

gentleMACS™/Extract-N-Amp™.

In this method, pericarp tissue was again placed directly onto the rotorof a gentleMACS™ M tube. 300 μL of sterile water was added to the tube,which was then closed and placed in a gentleMACS™ machine. The automatedprogram “Protein_01.01” was run. For pericarp tissues that were notfully dissociated, further mixing and running of the automated programwas done. The homogenate was transferred to a 1.5 mL microcentrifugetube and centrifuged for 1 minute at 10,000 rpm. The supernatant wasremoved without disturbing the tissue pellet at the bottom of the tube.30 μL of Extraction Solution/Seed Preparation Solution mix(Sigma-Aldrich Extract-N-Amp™ Seed PCR kit) was added and the resultingmixture was thoroughly mixed. The mixture was transferred to PCR striptubes for use on the thermocycler, which was programmed to hold 55° C.for 10 minutes, then 95° C. for 3 minutes, then to hold 4° C.indefinitely. 30 μL of Neutralization Solution B was added.

Liquid Nitrogen/SBEADEX®:

1.5 mL microcentrifuge tube pestles were placed in liquid nitrogen tocool. Pericarp tissue was placed in microcentrifuge tubes along with thecooled pestles and the entire tube was placed in liquid nitrogen. Liquidnitrogen was added to the tubes. The pericarp tissue was ground slowlyand thoroughly using the pestle. The tubes were occasionally dipped backinto the liquid nitrogen to keep the tissue cold. After grinding, 90 μLof Lysis buffer PN was added to each tube, which was then brieflycentrifuged then incubated at 65° C. for 1 hour. 120 μL of bindingbuffer PN and 10 μL of SBEADEX® particles were added to fresh tubes, andthe lysate from the grinding step was added to the new tubes. These werethen incubated at room temperature for 4 minutes to allow the DNA tobind to the SBEADEX® particles. The mixtures were then briefly vortexedand placed in a magnetic rack to concentrate the beads. The lysis bufferwas removed and 200 μL of wash buffer PN1 was added to each tube and thebeads were resuspended. The tubes were again placed in a magnetic rackto concentrate the beads and the wash buffer PN1 was removed. Thiswashing procedure was repeated using 200 μL of wash buffer PN2, thenrepeated again using 200 μL of pure water. Following this third washingstep, 20 μL of elution buffer PN was added and the tubes were incubatedat 55° C. for 10 minutes and vortexed every 3 minutes. A magnetic platewas used to concentrate the beads, and the eluate was transferred intofresh tubes, then stored at −20° C. until molecular characterization.

Extract-N-Amp™.

A master mix of 18 parts extraction solution and 2 parts of seedpreparation solution was made and 20 μL of the solution added topericarp tissue in 0.2 mL PCR strip tubes. The mixtures were placed in athermocycler set at 55° C. for 10 minutes, 95° C. for 3 minutes, then 4°C. indefinitely. 20.0 μL of Neutralization Solution B was added and theliquid portion of the mixture was transferred to fresh 1.5 mLmicrocentrifuge tubes.

D. Molecular Testing

QUBIT® dsDNA HS Assay Kit:

QUBIT® reagent was diluted into QUBIT® buffer at a 1:200 ratio to make aworking solution. 1 μL of the PCR products of step 2B was transferred to0.5 mL QUBIT® assay tubes and 199 μL of the working solution. Standardswere made by adding 10 μL of standard to 190 μL of QUBIT® workingsolution. The PCR products and standards were vortexed for 2-3 secondsthen briefly centrifuged. The tubes were then incubated at roomtemperature for 2 minutes. The tubes were then inserted into a QUBIT®2.0 fluorometer and readings were recorded.

Whole Genome Amplification (Seqplex):

The preferred method of whole genome amplification is the Seqplex methodusing the Seqplex Enhanced DNA Amplification Kit. To 1 μL of each DNAsolution generated in step C was added 2 μL library preparation bufferand 11 μL pure water. The solution was centrifuged, vortexed, andcentrifuged again, incubated on a thermocycler at 95° C. for 2 minutes,then held at 4° C. After cooling, 1 μL of library preparation enzyme wasadded. The solution was centrifuged, vortexed, and centrifuged again,then incubated on a thermocycler at 16° C. for 20 minutes, 24° C. for 20minutes, 37° C. for 20 minutes, 75° C. for 5 minutes, then held at 4° C.The solution was the briefly centrifuged. 15 μL of this solution wasadded to 15 μL of 5× Amplification Mix (A5112), 1.5 μL DNA Polymerasefor SeqPlex (SP300), 42.5 μL sterile water (W4502) and 1 μL SYBR Green(S9403), diluted 1:1000. This solution was mixed thoroughly, and eachreaction mix was divided into five 15 μL aliquots on a 384 well plate.The amplification thermocycle began with an initial denaturation at 94°C. for 2 minutes followed a sufficient number of cycles to reach 2-3cycles into the plateau (typically about 24 cycles): 94° C. denature for15 seconds, 70° C. anneal/extend for 5 minutes, read fluorescence,repeat. After cycling, the reaction mix was held at 70° C. for 30minutes then held at 4° C. After cooling, the samples were purified viaQIAquick PCR purification.

Whole Genome Amplification (REPLI-g Single Cell Kit):

Denaturation buffer D1 was prepared by adding 3.5 μL of reconstitutedbuffer DLB and 12.5 nuclease-free water. Neutralization buffer N1 wasprepared by adding 4.5 μL of stop solution and 25.5 μL of nuclease-freewater. 2.5 μL of the denaturation buffer was added to each 2.5 μLaliquot of DNA solution prepared in step C. This solution was incubatedat room temperature for 3 minutes. 5.0 μL of the neutralization bufferN1 was added, and the solution was vortexed then centrifuged briefly.Master mix was prepared with 9.0 μL nuclease-free water, 29.0 μL ofREPLI-g reaction buffer, and 2.0 μL of REPLI-g DNA polymerase perreaction. 40.0 μL of this master mix was added to each solution, whichis then run on a thermocycler at 30° C. for 8 hours, then cooled to 4°C.

The whole genome amplification products were evaluated using the QUBIT®assay to determine yield of DNA.

Genotyping Assays.

Both high density markers (the ILLUMINA® 3072X chip) and Taqman markeranalysis were successfully employed to genotype the genetic materialsdescribed in this example. Data demonstrating the effectiveness of theforegoing techniques is presented in FIGS. 2-4. FIG. 15 compares thedata quality obtained using DNA extraction methods against that obtainedusing whole genome amplification. While both methods give acceptableresults, the whole genome amplification method gives preferable results,with each of the three haplotypes well-resolved. FIG. 16 is afluorescent marker scatter plot demonstrating that quality fluorescentmarker data can be obtained from a single pericarp tissue sample. Infact, the methods of the invention allow genotyping using many markers,tens or potentially hundreds, using pericarp tissue extracted from asingle seed. FIG. 17 demonstrates the reliability of the methods of theinvention because of the high degree of similarity between the measuredgenotype of the pericarp tissue extracted from a single seed (each line)and the known maternal genotype.

We claim:
 1. A method of selecting maize embryonic tissue to cultureinto a plant with a known genotype comprising: (a) collecting shedcellular material from maize embryonic tissue in a container comprisingthe maize embryonic tissue and a non-destructive medium consistingessentially of an aqueous solution, wherein the shed cellular materialwas obtained by agitating the container without the addition ofchemicals that would induce cell lysis; (b) removing the maize embryonictissue from the container comprising the shed cellular material and thenon-destructive medium; (c) obtaining DNA from the shed cellularmaterial in the non-destructive medium consisting essentially of anaqueous solution; (d) genotyping the DNA from the shed cellularmaterial; (e) selecting maize embryonic tissue to culture into a plantbased on the genotype of the DNA from the shed cellular material; and(f) culturing the maize embryonic tissue to produce a maize plant with aknown genotype.
 2. The method of claim 1, wherein the maize embryonictissue comprises diploid tissue.
 3. The method of claim 1, wherein themaize embryonic tissue comprises haploid tissue.
 4. The method of claim1, wherein the maize embryonic tissue comprises doubled haploid tissue.5. The method of claim 1, wherein the DNA is obtained by exposing theshed cellular material to cold and then heat followed by agitation. 6.The method of claim 5, wherein said cold, heat, and agitation steps arerepeated.
 7. The method of claim 1 wherein the DNA is obtained byheating of the shed cellular material and agitation.
 8. The method ofclaim 7 wherein said heat and agitation steps are repeated.
 9. Themethod of claim 1, wherein obtaining DNA from the shed cellular materialcomprises extracting the DNA by adding magnetic particles to the shedcellular material.
 10. The method of claim 1, wherein said genotypingcomprises one or more of single nucleotide polymorphism detection,restriction fragment length polymorphism identification, randomamplified polymorphic detection, amplified fragment length polymorphismdetection, DNA sequencing, whole genome sequencing, allele specificoligonucleotide probes, and DNA hybridization to DNA microarrays orbeads.
 11. The method of claim 1, further comprising performing wholegenome amplification on the DNA from the shed cellular material.
 12. Themethod of claim 1, wherein the maize embryonic tissue has been removedfrom the container using an automated method.
 13. The method of claim 1,wherein the non-destructive medium consisting essentially of an aqueoussolution also comprises a chromosomal doubling agent.
 14. The method ofclaim 13, wherein the chromosomal doubling agent is colchicine.
 15. Themethod of claim 1, wherein the maize embryonic tissue is stored at 4° C.16. The method of claim 1, wherein the maize embryonic tissue is storedto prevent germination until after the genotype of the DNA from the shedcellular material has been obtained.